Multidrug-resistant Corynebacterium diphtheriae in people with travel history from West Africa to France, March to September 2023

We describe 10 unlinked cases of Corynebacterium diphtheriae infection (nine cutaneous, one respiratory) in France in 2023 in persons travelling from Guinea, Mali, Senegal, Niger or Nigeria and Central African Republic. Four isolates were toxigenic. Seven genomically unrelated isolates were multidrug-resistant, including a toxigenic respiratory isolate with high-level resistance to macrolides and beta-lactams. The high rates of resistance, including against first-line agents, call for further microbiological investigations to guide clinical management and public health response in ongoing West African outbreaks.

While diphtheria is largely controlled in high-income countries, regions with suboptimal vaccination coverage can experience large outbreaks, and travel from endemic or epidemic regions may result in case importation [1][2][3][4].Whereas classical diphtheria presents as respiratory infection caused by Corynebacterium diphtheriae, cutaneous infections are common and play an important role in transmission [5].Here we report on 10 unlinked cases of Corynebacterium diphtheriae infection (nine cutaneous, one respiratory) in France in 2023 in persons travelling from Guinea, Mali, Senegal, Niger or Nigeria and Central African Republic.

Diphtheria surveillance at the national reference laboratory
In France, diphtheria is a mandatory notifiable disease and isolates are submitted to the national reference laboratory (NRL) at Institut Pasteur, Paris together with information on sample type, demographics and travel history of the patients and clinical presentation.Diagnosis at the NRL relies on a multiplex quantitative PCR (qPCR) assay for detection of the tox gene coding for diphtheria toxin and for identification of C. diphtheriae and C. ulcerans [6].Production of the toxin is determined from isolated colonies using Elek's immunoprecipitation test [7].

Isolates submitted to the national reference laboratory between March and September 2023
Between 30 March and 30 September 2023, the NRL received and analysed 12 C. diphtheriae isolates from 12 cases with a travel history in West Africa (11 isolates) and Central African Republic (one isolate) (Table 1).Time between entry into France and sampling ranged from 5 to 31 days.All isolates had been identified as C. diphtheriae by MALDI-TOF mass spectrometry (Bruker, Germany) before being sent to the NRL, where identification was confirmed by qPCR.Three isolates were linked in a single household and had the same genome sequence, only the index case was included in further analysis.The 10 deduplicated isolates were characterised using biovar determination, antimicrobial susceptibility testing (AST) and genomic sequencing (Nextera genomic library and Illumina NextSeq-500 chemistry) as described [8,9].Methods for AST and interpretation of the results followed the European Committee on Antimicrobial Susceptibility Testing (EUCAST) 2023 recommendations [10] and for some of them Antibiogram Committee of the French Society of Microbiology recommendations [11,12] (Table 2).

Demographic and clinical characteristics of cases
All isolates except one were from cutaneous infections.Four cases had cutaneous infections in leg wounds and one had a Buruli ulcer.Four isolates were tox-positive by qPCR and produced the toxin phenotypically, as demonstrated by Elek's test.Two cases, infected with tox-positive strains, were hospitalised.No deaths were reported.Cases seeked healthcare in six French administrative districts ('Régions') and were aged 1-67

Table 1a
Characteristics of Corynebacterium diphtheriae isolates from people with travel history to West Africa, France, March-September 2023 (n = 10) a

Characteristics
Isolate identification number

Table 1b
Characteristics of Corynebacterium diphtheriae isolates from people with travel history to West Africa, France, March-September 2023 (n = 10) a years (median 21).Sex-ratio was 1:1.A recent travel history from Guinea (n = 3), Mali (n = 3), Senegal (n = 2), Central African Republic (n = 1) and Niger and Nigeria (n = 1) was documented.For six cases, vaccination status was unknown, three were not vaccinated and only one (aged 1 year) was vaccinated.

Multilocus sequence types
The 10 deduplicated isolates corresponded to nine multilocus sequence typing (MLST) sequence types, as ST377 was found in two cases, linked to Central African Republic and Guinea (Table 1).However, these two isolates belonged to two distinct genomic clusters by core genome MLST (Table 1).The high level of genomic diversity showed that the 10 isolates were not closely related phylogenetically and highlighted a multiclonal bacterial population.All isolates were biochemically characterised as biovar Mitis except the respiratory isolate, which was biovar Gravis, consistent with the presence in its genome of the spuA gene (Table 1).

Antimicrobial susceptibility testing
Antimicrobial susceptibility testing showed resistance against most antimicrobial agents (Figure 1A).Half or more of the isolates were resistant to folate pathway inhibitors, gentamicin, tetracycline and, more worryingly, benzylpenicillin.However, all except one were susceptible to amoxicillin, the recommended antimicrobial in France.Defining multidrug resistance as resistance to agents in three or more classes (discarding fosfomycin, to which Corynebacterium species are intrinsically resistant), seven of the 10 isolates were multidrug-resistant.The 10 isolates were resistant to 3-12 individual agents (Figure 1B), considering the 21 antimicrobial agents tested (excluding fosfomycin).Five isolates were resistant to benzylpenicillin and two of these were additionally resistant to erythromycin (three to azithromycin), i.e. to both first-line recommended treatments.
isolate (FRC1984, linked to Guinea) had high-level resistance to benzylpenicillin and oxacillin (minimum inhibitory concentration (MIC) > 256 mg/L), amoxicillin (MIC 64 mg/L), erythromycin (MIC 16 mg/L) and azithromycin (MIC > 256 mg/L).Tetracycline and rifampicin were also inactive against this isolate, which, however, remained susceptible at increased exposure to ciprofloxacin.Surprisingly, the isolate was also resistant to meropenem (MIC 4 mg/L).However, we noted that this isolate presented a heterogeneous resistance to penicillin, with a characteristic halo denoting higher susceptibility in parts of the bacterial population (Figure 2A).High-level resistance to beta-lactams and carbapenems, associated with heteroresistance, was previously reported only once, to our knowledge [13].
Genomic sequencing disclosed antimicrobial resistance genes or mutations that were highly consistent with phenotypes (Table 1).Gene pbp2m (also named pbp2C by Forde et al. 2021 [13]) was found in five isolates, all showing low-level resistance to benzylpenicillin (MIC 1.5 or 2 mg/L) except isolate FRC1984 (see above).The macrolide-resistance gene ermX was found in the three macrolide-resistant isolates (travel from Guinea, Mali and Niger), always co-located with pbp2m.This physical association was previously reported [8].The heteroresistance observed to erythromycin in addition to penicillin can thus be explained by the co-localisation of pbp2m and ermX on a single genetic element, which may have been amplified as described previously [13].

Discussion
In 2023, outbreaks of diphtheria were declared in several West African countries.Nigeria has been experiencing a large outbreak, with 13,416 suspected cases since 9 May 2022 [14][15][16].Guinea has also declared an outbreak that started in early July 2023, with 538 cases by 13 October [17].Cases have also been reported in Mali and Niger [18].A low diphtheria vaccination rate, exacerbated following disruption of public health prevention and response due to COVID-19 pandemic, is an important driver of diphtheria outbreaks.In Nigeria, widespread resistance to benzylpenicillin, ciprofloxacin and trimethoprim-sulfamethoxazole was reported [14], but the levels and mechanisms of resistance remain so far unknown.A possible genetic link between outbreak strains in different West African countries has not been investigated.
Despite nearly one year from the outbreak declarations in West Africa [15], little information is available on the circulating isolates and their genomic or phenotypic characteristics.Travel-related cases from West Africa to metropolitan France in 2023 may provide a window on the ongoing outbreaks.Here, we showed that eight of 10 unlinked isolates were resistant to multiple antimicrobial agents.Gene pbp2m typically confers low-level resistance to penicillin and amoxicillin [8].In our previous global genomic survey, we reported pbp2m in less than 5% of non-duplicate C. diphtheriae isolates [9].Here, six of 10 isolates were carrying pbp2m, a remarkable observation given that these isolates belong to ten distinct genomedefined types.This surprising result suggests a high prevalence of pbp2m in West Africa and might even indicate an African origin of pbp2m in C. diphtheriae.Gene ermX also appeared to be common in West Africa and was always associated with pbp2m herein.Mobile elements carrying both pbp2m and ermX should be the object of enhanced surveillance and further research, especially given their possible genetic amplification upon antimicrobial exposure.
Multidrug resistance is of concern, as it affects negatively clinical care and prophylactic effectiveness to control further disease and spread.Here, resistance was observed both in tox-positive and tox-negative isolates.The latter isolates were detected by taking samples from cutaneous infections and using mass spectrometry technology to identify bacterial colonies isolated from skin lesions, i.e. not only from classical respiratory diphtheria.Although cutaneous C. diphtheriae infections are not typically detected in outbreak settings where diagnosis is mainly based on classical clinical signs of respiratory diphtheria, infected wounds are considered as important for transmission of diphtheria [5].
The data presented here may be of relevance for the ongoing resurgence of diphtheria in West Africa.However, a limitation of this study is the small number of C. diphtheriae isolates and uncertainty whether they are representative of the main genotypes circulating in West Africa.It is urgent that more isolates, representative of the ongoing outbreaks, are characterised to guide appropriate antimicrobial therapy and prophylaxis strategies.In addition to representing a potential health threat to West African populations, circulation of multidrug-resistant C. diphtheriae is of broader concern and underlines the need to maintain high vaccination coverage even in low-incidence regions, which face the risk of case importation.Despite this, the risk of dissemination within high-vaccination countries seems limited.In France in 2023, no secondary cases were reported, as also observed for cases imported from other areas in 2022 [9].

Conclusion
Diphtheria, a largely forgotten disease, seems to be re-emerging predominantly in vulnerable populations with low vaccination rates.Furthermore, it presents an additional risk due to the emergence of multidrugresistant strains.There is an urgent need to reinforce diagnostic laboratory capacity for diphtheria to guide clinical and public health response and to implement microbiological methods including genomic sequencing to decipher transmission patterns within countries, genetic support for resistance, and the links between outbreaks at regional scale.

Ethical statement
Diphtheria is a notifiable disease in France.Phenotypic and genotypic analyses of bacterial isolates were carried out within the framework of the mandate given to the National Reference Center for Corynebacteria of the Diphtheriae Complex the Ministry of Health (Public Health France).All French bacteriological samples and associated clinical data are collected, coded, shipped, managed and analysed according to the French National Reference Center protocols that received approval by French supervisory ethics authority (CNIL, n°1474671).
inhibitory concentration; MLST: multilocus sequence typing; NA: not applicable; SIR: susceptible, intermediate, resistant; ST: sequence type.a Three isolates were linked in a single household and had the same genome sequence, only the index isolate was included in further analysis.Therefore, 10 isolates were included in the analysis and not 12.b Time between entry into France and sampling ranged from 5 to 31 days.c Corynebacterium diphtheriae biovar Gravis marker.

Figure 1
Figure 1Phenotypic resistance and multidrug resistance of Corynebacterium diphtheriae isolates from patients with travel history to West Africa, France, 2023 (n = 10)

Table 2
Antimicrobial agents used and breakpoints applied for testing Corynebacterium diphtheriae isolates from people with travel history to West Africa, France, 2023 (n = 10)